Peronospora resistance in spinacia sp

ABSTRACT

The invention pertains to spinach plants comprising a gene or locus which leads to a broad spectrum resistance to  Peronospora farinosa  f sp.  spinaciae  (Pfs). The invention also relates to progeny of said spinach plants, to propagation material of said spinach plants, to a cell of said spinach plants, to seed of said spinach plants, and to harvested leaves of said spinach plants. This invention further relates to use of said spinach plants in breeding to confer resistance against  Peronospora farinosa  f. sp.  spinaciae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/EP2016/077320, filed Nov. 10, 2016, International ApplicationNo. PCT/EP2016/077322, filed Nov. 10, 2016, U.S. application Ser. No.14/937,696, filed Nov. 10, 2015, and U.S. application Ser. No.14/937,714, filed Nov. 10, 2015, the content of each of which isincorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 781982000800SEQLIST.txt,date recorded: May 8, 2018, size: 3 KB).

FIELD

The invention pertains to spinach plants comprising a gene or locuswhich leads to a broad spectrum resistance to Peronospora farinosa f.sp. spinaciae (Pfs). The invention also relates to progeny of saidspinach plants, to propagation material of said spinach plants, to acell of said spinach plants, to seed of said spinach plants, and toharvested leaves of said spinach plants. This invention further relatesto use of said spinach plants in breeding to confer resistance againstPeronospora farinosa f. sp. spinaciae.

BACKGROUND

Spinach (Spinacia oleracea) is a flowering plant from the Amaranthaceaefamily that is grown as a vegetable. The consumable parts of spinach arethe leaves from the vegetative stage. Spinach is sold loose, bunched, inprepacked bags, canned, or frozen. There are three basic types ofspinach, namely the savoy, semi-savoy and smooth types. Savoy hascrinkly and curly leaves. Flat or smooth leaf spinach has in generalbroad, smooth leaves. Semi-savoy is a variety with slightly crinkledleaves. The main market for spinach is baby-leaf. Baby spinach leavesare usually of the flat-leaf variety and usually the harvested leavesare not longer than about eight centimeters. These tender, sweet leavesare sold loose rather than in bunch. They are often used in salads, butcan also be lightly cooked. Downy mildew, which in spinach is caused bythe pathogen Peronospora farinosa f. sp. spinaciae (formerly known as P.effusa), is a major threat for spinach growers, because it affects theharvested plant parts, namely the leaves. Infection makes the leavesunsuitable for sale and consumption, as it manifests itselfphenotypically as yellow lesions on the older leaves, and on the abaxialleaf surface a greyish fungal growth can be observed. The infection canspread very rapidly, and it can occur both in glasshouse cultivation andin soil cultivation. The optimal temperature for formation andgermination of P. farinosa f. sp. spinaciae is 9 to 12° C., and it isfacilitated by a high relative humidity. When pathogens are deposited ona humid leaf surface they can readily germinate and infect the leaf.Pathogen growth is optimal between 8 and 20° C. and a relative humidityof ≥80%, and growth can be observed within 6 and 13 days afterinfection. P. farinosa can survive in the soil for up to 3 years, or inseeds or living plants. In recent years various resistance genes havebeen identified that provide spinach plants with a resistance againstdowny mildew. However, it has been observed that previously resistantspinach cultivars can again become susceptible to the pathogen.Investigations revealed that the cultivars themselves had not changed,and that the loss of downy mildew resistance must therefore be due to P.farinosa overcoming the resistance in these spinach cultivars. The downymildew races that were able to infect resistant spinach cultivars havebeen identified on a differential reference set, used to test spinachcultivars for resistance. The differential set comprises a series ofspinach cultivars (hybrids) that have different resistance patterns tothe currently identified pathogenic races. To date, 17 pathogenic racesof spinach downy mildew (Pfs) have been officially identified andcharacterized. Races 4 through 10 were identified between 1990 and 2009,which illustrates the versatility and adaptability of the pathogen toovercome resistances in spinach. In 2014, isolate UA1014APLP (also knownas UA1014 and now Pfs 17) was identified by the Correll lab of Arkansas.

In different geographical regions different combinations of pathogenicraces or isolates occur, and the spinach industry therefore has a strongdemand for spinach cultivars that are resistant to as many relevantdowny mildew races as possible, preferably to all races that may occurin their region, and even to the newest threats that cannot be counteredwith the resistances that are present in the commercially availablespinach cultivars.

It is crucial to stay at the forefront of developments in this field, asPeronospora continuously develops the ability to break the resistancesthat are present in commercial spinach varieties. For this reason newresistance genes are very valuable assets, and they form an importantresearch focus in spinach breeding. The goal of spinach breeders is torapidly develop spinach varieties with resistance to as many Peronosporaraces as possible, including the latest identified. To date, 17 Pfsraces are officially recognized and made publicly available from theDepartment of Plant Pathology, University of Arkansas, Fayetteville,Ark. 72701, USA, and also from NAK Tuinbouw, Sotaweg 22, 2371 GDRoelofarendsveen, the Netherlands. Pfs 15 was officially recognized in2014 by the International Working Group on Peronospora farinosa (IWGP).Recently identified isolate UA1014APLP is yet to be officiallyrecognized as a race, but is already of importance to breeders.

It is the object of the invention to provide spinach plants, conferringresistance to various Peronospora races and/or isolates, including theones that have been most recently identified.

BRIEF SUMMARY

The present invention provides spinach plants comprising resistanceagainst one or more of Peronospora farinosa races 1, 2, 3, 4, 5, 6, 7,8, 9, 11, 12, 13, 14, 15, and isolate UA1014APLP (Pfs 17). Said plantsare obtainable by introgression from a plant grown from seeds of whichrepresentative samples have been deposited with the National Collectionof Industrial, Food and Marine Bacteria (NCIMB) Ltd. under accessionnumbers NCIMB 42392 and NCIMB 42393. The plants have a broad spectrum ofPeronospora farinosa resistance, which is stably transferred to theprogeny.

Definitions

The indefinite article “a” or “an” does not exclude the possibility thatmore than one of the element is present, unless the context clearlyrequires that there be one and only one of the elements. The indefinitearticle “a” or “an” thus usually means “at least one”.

“Plant variety” is a group of plants within the same botanical taxon ofthe lowest grade known, which (irrespective of whether the conditionsfor the recognition of plant breeder's rights are fulfilled or not) canbe defined on the basis of the expression of characteristics that resultfrom a certain genotype or a combination of genotypes, can bedistinguished from any other group of plants by the expression of atleast one of those characteristics, and can be regarded as an entity,because it can be multiplied without any change. Therefore, the term“plant variety” cannot be used to denote a group of plants, even if theyare of the same kind, if they are all characterized by the presence ofone or two loci or genes (or phenotypic characteristics due to thesespecific loci or genes), but which can otherwise differ from one anotherenormously as regards the other loci or genes.

“Spinach” or “cultivated spinach” or “cultivated Spinacia oleracea”refers herein to plants of the species Spinacia oleracea (or seeds fromwhich the plants can be grown), and parts of such plants, bred by humansfor food and having good agronomic characteristics. This includes anycultivated spinach, such as breeding lines (e.g. backcross lines, inbredlines), cultivars and varieties (open pollinated or hybrids). Thisincludes any type of spinach, such as savoy, flat- or smooth-leafspinach or semi-savoy types. Wild spinach (i.e. not cultivated spinach)or wild relatives of spinach, such as Spinacia tetrandra and Spinaciaturkestanica, are not encompassed by this definition.

As used herein, the term “plant” includes the seed (from which the plantcan be grown), the whole plant or any parts such as plant organs (e.g.,harvested or non-harvested leaves, etc.), plant cells, plantprotoplasts, plant cell- or tissue cultures from which whole plants canbe regenerated, propagating or non-propagating plant cells, plants cellswhich are not in tissue culture (but which are for example in vivo in aplant or plant part), plant callus, plant cell clumps, planttransplants, seedlings, plant cells that are intact in plants, plantclones or micro-propagations, or parts of plants (e.g., harvestedtissues or organs), such as plant cuttings, vegetative propagations,embryos, pollen, ovules, flowers, leaves, heads, seeds (produced on theplant after self-fertilization or cross-fertilization), clonallypropagated plants, roots, stems, stalks, root tips, grafts, parts of anyof these and the like, or derivatives thereof, preferably having thesame genetic make-up (or very similar genetic make-up) as the plant fromwhich it is obtained. Also any developmental stage is included, such asseedlings, cuttings prior or after rooting, mature and/or immatureplants or mature and/or immature leaves. When “seeds of a plant” arereferred to, these either refer to seeds from which the plant can begrown or to seeds produced on the plant, after self-fertilization orcross-fertilization.

“Somatic cells” and “reproductive cells” can be distinguished, wherebysomatic cells are cells other than gametes (e.g. ovules and pollen),germ cells and gametocytes. Gametes, germ cells and gametocytes are“reproductive cells.

“Tissue Culture” or “cell culture” refers to an in vitro compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into plant tissue. Tissue culturesand cell cultures of spinach, and regeneration of spinach plantstherefrom, is well known and widely published (see, e.g. Nguyen et al.,2013, Plant Biotechnology Reports, Vol. 7 Issue 1, p 99).

“Harvested plant material” refers herein to plant parts (e.g., leavesdetached from the whole plant) which have been collected for furtherstorage and/or further use.

“Harvested seeds” refers to seeds harvested from a line or variety,e.g., produced after self-fertilization or cross-fertilization andcollected.

“Harvested leaves” as used herein refers to spinach leaves, i.e., theplant without the root system, for example substantially all (harvested)leaves.

“Progeny” or “progenies” or “descendants” as used herein refers tooffspring, or the first and all further descendants derived from(obtainable from) (derivable from or obtained from) a plant of theinvention that comprises (retains) the resistance gene in homozygous orheterozygous form and/or the resistance phenotype described herein.Progeny may be derived by regeneration of cell culture or tissueculture, or parts of a plant, or selfing of a plant, or by producingseeds of a plant. In further embodiments, progeny may also encompassspinach plants derived from crossing of at least one spinach plant withanother spinach plant of the same or another variety or (breeding) line,and/or backcrossing, and/or inserting of a locus into a plant and/ormutation. A progeny is, e.g., a first generation progeny, i.e. theprogeny is directly derived from, obtained from, obtainable from orderivable from the parent plant by, e.g., traditional breeding methods(selfing and/or crossing) or regeneration. However, the term “progeny”generally encompasses further generations such as second, third, fourth,fifth, sixth, seventh or more generations, i.e., generations of plantswhich are derived from, obtained from, obtainable from or derivable fromthe former generation by, e.g., traditional breeding methods,regeneration or genetic transformation techniques. For example, a secondgeneration progeny can be produced from a first generation progeny byany of the methods mentioned above. Also double haploid plants areprogeny.

“Plant line” is for example a breeding line which can be used to developone or more varieties.

“F1, F2, F3, etc.” refers to the consecutive related generationsfollowing a cross between two parent plants or parent lines. The plantsgrown from the seeds produced by crossing two plants or lines is calledthe F1 generation. Selfing the F1 plants results in the F2 generation,etc.

“Hybrid” refers to the seeds harvested from crossing one plant line orvariety with another plant line or variety, and the plants or plantparts grown from said seeds.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two non-isogenic inbred parent lines. Thus, F1 hybrid seeds areseeds from which F1 hybrid plants grow.

An “interspecific hybrid” refers to a hybrid produced from crossing aplant of one species (e.g., S. oleracea) with a plant of another species(e.g., S. tetrandra or S. turkestanica).

“Crossing” refers to the mating of two parent plants. Equally“Cross-pollination” refers to fertilization by the union of two gametesfrom different plants.

“Selfing” refers to the self-pollination of a plant, i.e. to the unionof gametes from the same plant.

“Backcrossing” refers to a breeding method by which a (single) trait,such as Pfs resistance conferred by a resistance gene, can betransferred from one genetic background (also referred to as “donor”;generally but not necessarily this is an inferior genetic background)into another genetic background (also referred to as “recurrent parent”;generally but not necessarily this is a superior genetic background). Anoffspring of a cross (e.g. an F1 plant obtained by crossing a wildspinach or wild relative of spinach with a cultivated spinach; or an F2plant or F3 plant, etc., obtained from selfing the F1) is “backcrossed”to the parent with the superior genetic background, e.g. to thecultivated parent.

After repeated backcrossing, the trait of the donor genetic background,e.g. the resistance gene, will have been incorporated into the recurrentgenetic background. The terms “gene converted” or “conversion plant” or“single locus conversion” in this context refer to plants which aredeveloped by backcrossing wherein essentially all of the desiredmorphological and/or physiological characteristics of the recurrentparent are recovered in addition to the one or more genes (e.g. theresistance gene) transferred from the donor parent.

The term “traditional breeding techniques” encompasses herein crossing,backcrossing, selfing, selection, chromosome doubling, double haploidproduction, embryo rescue, the use of bridge species, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,the − resistance gene can be obtained, identified, selected, and/ortransferred.

“Regeneration” refers to the development of a plant from in vitro cellculture or tissue culture or vegetative propagation.

“Vegetative propagation”, “vegetative reproduction” or “clonalpropagation” are used interchangeably herein and mean the method oftaking part of a plant and allowing that plant part to form at leastroots where plant part is, e.g., defined as or derived from (e.g. bycutting off) leaf, pollen, embryo, cotyledon, hypocotyl, cells,protoplasts, meristematic cell, root, root tip, pistil, anther, flower,shoot tip, shoot, stem, fruit, and petiole. When a whole plant isregenerated by vegetative propagation, it is also referred to as a“vegetative propagation” or a “vegetatively propagated plant”.

“Single locus converted (conversion) plant” refers to plants which aredeveloped by plant breeding techniques comprising or consisting ofbackcrossing, wherein essentially all of the desired morphologicaland/or physiological characteristics of a spinach plant are recovered inaddition to the characteristics of the single locus having beentransferred into the plant via the backcrossing technique and/or bygenetic transformation.

“Transgene” or “chimeric gene” refers to a genetic locus comprising aDNA sequence which has been introduced into the genome of a spinachplant by transformation. A plant comprising a transgene stablyintegrated into its genome is referred to as “transgenic plant”.

“Transgene” or “chimeric gene” refers to a genetic locus comprising aDNA sequence which has been introduced into the genome of a spinachplant by transformation. A plant comprising a transgene stablyintegrated into its genome is referred to as “transgenic plant”.

“Pfs” or “Peronospora farinosa” or “downy mildew” refers to races of thepathogen Peronospora farinosa f sp. spinaciae. Pfs 1-17 refer to theofficially recognized races, which can be differentiated on thedifferential hosts of spinach and which can be obtained from theNaktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands, orvia references provided by the ISF (International Seed Federation).

“Differential hosts” or “differentials” refers to the differential hostsof spinach for distinguishing Pfs 1-17, which can be obtained from theNaktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands, orvia references provided by the ISF (International Seed Federation).

A “Pfs resistant plant” or “downy mildew resistant plant” or a planthaving “Pfs resistance” or a “Pfs resistant phenotype” refers to aspinach plant which is resistant against one or more pathogenic racesand/or pathogenic isolates of Pfs, as determined in a qualitativeresistance assay under controlled environmental conditions. In such aresistance assay a plurality of plants (e.g. at least 2 replicates of atleast 10 plants) of a genotype, are inoculated with a sporangiasuspension of the race or isolate and incubated under suitableconditions. After a suitable incubation period (e.g. 7, 8, 9, 10, 11 ormore days after inoculation) the plants are evaluated for symptoms.Susceptible controls should show sporulation at the time of symptomevaluation. Any plant showing sporulation on the cotyledons (and/or onthe true leaf/leaves) is considered “susceptible”, while any plant notshowing any sporulation on the cotyledons (and/or on the trueleaf/leaves) is considered “resistant”. Additionally, any plants showingsparse sporulation on the tips of cotyledons (and/or on the trueleaf/leaves), indicative of a reduced level of infection, is considered“intermediate resistant.” A plant genotype with 95-100% of theinoculated plants being classified as “resistant” plants is consideredto be resistant against the race or isolate. In the test greaterthan >95% of inoculated plants (preferably 100% of plants) of thesusceptible control plant, such as cultivar ‘Viroflay’, should showsporulation. Suitable tests are described in Irish et al. 2007 (PlantDisease Vol. 91 No. 11, in Materials and Methods on page 1392-1394), orin Correll et al. 2010 (“Guidelines for Spinach Downy Mildew:Peronospora farinosa f sp. spinaciae (Pfs) found on the website of theInternational Seed Federation). As used herein, “+” refers tosusceptibility, “−” refers to resistance, and “(−)” refers tointermediate resistance, also known as field resistance.

The term “locus” (loci plural) means a specific place or places or asite on a chromosome where for example a gene or genetic marker isfound.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each chromosomeof the pair of homologous chromosomes. A diploid plant species maycomprise a large number of different alleles at a particular locus.These may be identical alleles of the gene (homozygous) or two differentalleles (heterozygous).

The term “gene” means a (genomic) DNA sequence comprising a region(transcribed region), which is transcribed into a messenger RNA molecule(mRNA) in a cell, and an operably linked to regulatory region (e.g. apromoter). Different alleles of a gene are thus different alternativeforms of the gene, which may be in the form of e.g. differences in oneor more nucleotides of the genomic DNA sequence (e.g. in the promotersequence, the exon sequences, intron sequences, etc.), mRNA and/or aminoacid sequence of the encoded protein.

“Allelism test” refers to a genetic test whereby it can be testedwhether a phenotype, such as Pfs resistance, seen in two plants, isdetermined by the same gene or by different genes. For example, theplants to be tested are crossed with each other, the F1 is selfed andthe segregation of the phenotypes amongst the F2 progeny is determined.Other segregating populations can equally be made (e.g. backcrosspopulations). The ratio of segregation of the phenotype indicates if thegenes are allelic (alleles of the same gene) or non-allelic (different,independent genes).

“Introgression fragment” or “introgression segment” or “introgressionregion” refers to a chromosome fragment (or chromosome part or region)which has been introduced into another plant of the same or relatedspecies by crossing or traditional breeding techniques, such asbackcrossing, i.e. the introgressed fragment is the result of breedingmethods referred to by the verb “to introgress” (such as backcrossing).In spinach, wild spinach or wild relatives of spinach are often used tointrogress fragments of the wild genome into the genome of cultivatedspinach. Such a spinach plant thus has a “genome of Spinacia oleracea”,but comprises in the genome a fragment of a wild spinach or spinachrelative. It is understood that the term “introgression fragment” neverincludes a whole chromosome, but only a part of a chromosome. Theintrogression fragment can be large, e.g. even half of a chromosome, butis preferably smaller, such as about 15 Mb or less, such as about 10 Mbor less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less,about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mbor less, about 2 Mb or less, about 1 Mb (equals 1,000,000 base pairs) orless, or about 0.5 Mb (equals 500,000 base pairs) or less, such as about200,000 bp (equals 200 kilo base pairs) or less, about 100,000 bp (100kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) orless.

“Physical distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is the actualphysical distance expressed in base pairs (bp), kilobase pairs (kb) ormegabase pairs (Mb).

“Genetic distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is measured byfrequency of crossing-over, or recombination frequency (RF) and isindicated in centimorgans (cM). One cM corresponds to a recombinationfrequency of 1%. If no recombinants can be found, the RF is zero and theloci are either extremely close together physically or they areidentical. The further apart two loci are, the higher the RF.

A genetic element, a locus, an introgression fragment or a gene orallele conferring a trait (such as resistance against Pfs) is said to be“obtainable from” or can be “obtained from” or “derivable from” or canbe “derived from” or “as present in” or “as found in” a plant or seed ifit can be transferred from the plant or seed in which it is present intoanother plant or seed in which it is not present (such as a line orvariety) using traditional breeding techniques without resulting in aphenotypic change of the recipient plant apart from the addition of thetrait conferred by the genetic element, locus, introgression fragment,gene or allele. The terms are used interchangeably and the geneticelement, locus, introgression fragment, gene or allele can thus betransferred into any other genetic background lacking the trait. Notonly seeds deposited and comprising the genetic element, locus,introgression fragment, gene or allele can be used, but alsoprogeny/descendants from such seeds which have been selected to retainthe genetic element, locus, introgression fragment, gene or allele, canbe used and are encompassed herein, such as commercial varietiesdeveloped from the deposited seeds or from descendants thereof. Whethera plant comprises the same genetic element, locus, introgressionfragment, gene or allele as obtainable from the deposited seeds can bedetermined by the skilled person using one or more techniques known inthe art, such as phenotypic assays, whole genome sequencing, molecularmarker analysis, trait mapping, chromosome painting, allelism tests andthe like.

The term “traditional breeding techniques” encompasses herein crossing,backcrossing, selfing, selection, chromosome doubling, double haploidproduction, embryo rescue, the use of bridge species, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,the resistance according to the current invention can be obtained,identified, selected, and/or transferred.

A “molecular marker”, “genetic marker”, or simply “marker” as usedherein, refers to a nucleotide sequence that contains, surrounds orassociates with variation (polymorphism) at a given genomic locus, andcan be used to identify plants having a particular allele. Molecularmarkers can be developed based on polymorphisms that include, but arenot limited to, single nucleotide polymorphism (SNP), insertion/deletion(InDel), simple sequence repeat (SSR), presence/absence variation (PAV),and copy number variation (CNV). Methods and techniques of developing,identifying and genotyping molecular markers are well known in the art.

A “single nucleotide variant” or “SNV” refers to a type of variant whereone nucleotide base is replaced by another nucleotide base. SNV issimilar in context with the more commonly used term “single nucleotidepolymorphism (SNP)”, but is a preferred term over SNP when noimplications of frequency in a population are involved. For the purposeof this application, the terms SNV and SNP are used interchangeably.

As used herein, a “genetic determinant” refers to the geneticinformation in the genome of a plant that causes or associates with atrait of interest. Genetic determinants include, but are not limited to,genes, alleles, genetic markers, and quantitative trait loci (QTL).

As used herein, the term “screening” refers to a process of evaluatingor identifying plant material for a property of interest. The propertyof interest can be a phenotypic property (e.g., disease resistance), ora genotypic property (e.g., presence of a certain allele).

DETAILED DESCRIPTION

The present invention concerns spinach plants comprising resistanceagainst one or more of Peronospora farinosa races 1, 2, 3, 4, 5, 6, 7,8, 9, 11, 12, 13, 14, 15, and isolate UA1014APLP (Pfs 17), preferablycomprising resistance against races Pfs 1, Pfs 2, Pfs 3, Pfs 4, Pfs 5,Pfs 6, Pfs 9, Pfs 11, Pfs 12, Pfs 13, Pfs 14 and Pfs 15, and Pfs 17. Theresistance is a broad range resistance, and may further compriseintermediate resistance to Pfs 7 and Pfs 8.

More specifically the plant is obtainable by (or obtained by, orderivable from, or derived from) introgression from a plant grown fromseeds of which a representative sample has been deposited with NCIMBunder accession number NCIMB 42392 or NCIMB 42393 or any plant derivedtherefrom.

The resistance trait may be inherited by a single gene, preferably adominant gene. In another embodiment, said resistance is conferred bytwo or more genes, or a multilocus. In fact, the resistance locus orloci (and the Pfs resistance phenotype conferred thereby), can betransferred from the seeds deposited under NCIMB 42392 or NCIMB 42393,or from progeny of said seeds, into any spinach line or variety bytraditional breeding techniques and can confer resistance against one ormore of Pfs races 1-9, 11-15 and isolate UA1014APLP (Pfs 17) resistance(and optionally resistance against new pathogenic isolates) onto anotherspinach plant. Thus, for example, a spinach plant of the invention canbe used as male or female parent in a cross with another spinach plant,and progeny, such as F1, F2, F3, or further generations of selfingand/or backcross progeny (e.g. BC1, BC2, BC1S1, BC2S1, BC1S2, etc.) canbe identified and selected, whereby the progeny comprise the same Pfsresistance phenotype as the initial plant of the invention. Selection ofprogeny for the presence of the resistance can, therefore, be carriedout using a disease resistance assay as described herein, wherebyresistance against one or more (or all) of the Pfs races is tested inthe progeny.

Whether a spinach plant genotype (i.e. a spinach line or variety)comprises resistance against one or more Pfs races or isolates can betested using qualitative disease resistance assays under controlledenvironment conditions. Different protocols of such assays exist and canbe used by the person skilled in the art. In short, seedlings of aplurality of plants of the plant genotype to be tested (e.g. at least 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) areinoculated with inoculum of the Pfs race and the seedlings are incubatedunder conditions which are favorable to the pathogen. Several days afterincubation, the plants are assessed for infection symptoms, especiallysporulation on the cotyledons and/or leaves (e.g. first true leaf), andeach plant is categorized as “resistant” (showing no signs ofsporulation) or “susceptible” (showing sporulation). If a certainpercentage of all plants of a genotype are classified as “resistant”,e.g. more than about 95%, 98%, 99% (or even 100%), then the spinachplant genotype is resistant to the race tested. Obviously, also one ormore control plants (e.g. a susceptible line or variety, a resistantline or variety) should be included in the assay using the sametreatment(s) and environmental conditions, to ensure that the assayworks as expected.

Alternatively or in addition to the phenotypic assay, selection oridentification of a spinach plant (e.g. a progeny plant) comprising theresistance gene or locus/loci of the invention may be achieved bydetecting one or more molecular markers linked to the resistance gene orlocus. This aspect will be described elsewhere herein.

In one of the embodiment of the invention, the spinach plant is aninbred line, especially an inbred line which can be used as a parent forF1 hybrid seed production. In another embodiment of the invention, thespinach plant is a hybrid, especially an F1 hybrid. An F1 hybrid may begenerated by crossing a first inbred parent line which comprises theresistance gene or locus/loci, preferably in homozygous form, with asecond inbred parent line. The first inbred parent line may be a linedeveloped from using seeds deposited under NCIMB 42392 or NCIMB 42393 orfrom progeny of plants grown from these seeds, whereby the progenyretain the Pfs resistance phenotype (and the resistance gene orlocus/loci).

The second inbred parent line may be any spinach line, i.e. it maycompletely lack Pfs resistance, or it may comprise a different Pfsresistance gene (and different resistance phenotype) or it may alsocomprise the resistance gene or locus/loci according to the currentinvention.

As mentioned, the spinach plant according to the invention may be anytype of spinach. For example, the spinach plant may be a savoy type, asemi-savoy type or flat- or smooth leaved spinach.

In other words, the resistance can be introduced into any other spinachplant by introgression from a plant grown from seeds of which arepresentative sample was deposited under NCIMB 42392 or NCIMB 42393, orany spinach plant derived therefrom and comprising the gene orlocus/loci. The deposited seeds are therefore a source of the resistanceof the invention, as are spinach plants not directly obtained from thedeposit, but for example indirectly obtained (e.g. later releasedcommercial varieties) and which contain the resistance gene orlocus/loci of the invention.

The resistance of the current invention was identified in wild materialfrom a genebank and was introduced through backcrossing into S.oleracea. In one aspect, therefore, a spinach plant is providedcomprising resistance against one or more of Pfs 1-9, 11-15 and isolateUA1014APLP (Pfs 17) and possibly new pathogenic isolates, wherein saidresistance against Peronospora farinosa is conferred by an introgressionfragment from wild spinach or from a wild relative of spinach. In apreferred aspect, a spinach plant is provided comprising resistanceagainst at least Pfs 1-6, 9, 11-15 and Pfs 17. The resistance is a broadrange resistance, and may further comprise intermediate resistance toPfs 7 and Pfs 8.

In one embodiment, the introgression fragment is the fragment as foundin (and as obtainable from; or obtained from; or derivable from; orderived from) spinach seeds, a representative sample of seeds havingbeen deposited under accession number NCIMB 42392 or NCIMB 42393. Thefragment can be identified by various methods, such as chromosomepainting or sequencing the spinach genome and identifying chromosomeparts which are introgressions from wild spinach or wild relatives ofspinach. The fragment can also be identified by one or more molecularmarkers (e.g. SNP markers, AFLP markers, RFLP markers, etc.), especiallymolecular markers which are polymorphic between cultivated spinach andthe wild introgression fragment.

In another embodiment, the introgression fragment is derived from thefragment as found in spinach seeds, a representative sample of seedshaving been deposited under accession number NCIMB 42392 or NCIMB 42393,whereby the introgression fragment is shorter but retains the resistancegene or locus/loci (and the Pfs resistance phenotype conferred by thegene). Spinach plants comprising such shorter introgression fragmentscan be generated by crossing a plant of the invention with anotherspinach plant and selecting recombinant progeny which retain theresistance phenotype conferred by the resistance gene or locus/loci, butwhich contain a shorter introgression fragment.

In one aspect a method is provided for generating spinach plantcomprising resistance against one or more of Pfs races 1-9, 11-15 andisolate UA1014APLP (Pfs 17), preferably at least Pfs races 1-6, 7(−),8(−), 9, 11-15 Pfs 17. The method comprises the steps of:

a) Providing a spinach plant comprising resistance against the desiredPfs races;b) Crossing said spinach plant with another spinach plant to produce F1seeds;c) Optionally selfing the plants grown from F1 seeds one or more timesto produce F2, F3 or further generation selfing progeny;d) Identifying (or selecting) spinach plants grown from F1, F2, F3 orfurther generation selfing progeny which have resistance against thedesired Pfs races;e) Optionally crossing said identified (or selected) F1 progeny orselfing progeny to the spinach plant of step b), to produce a backcrossprogeny;f) Optionally selecting backcross progeny comprising resistance againstthe desired Pfs races (e.g., preferably at least Pfs races 1-6, 7(−),8(−), 9, 11-15 and 17).

In another embodiment a method is provided for generating a spinachplant comprising resistance against one or more of Pfs 1-9, 11-15 andisolate UA1014APLP (Pfs 17), preferably at least Pfs races 1-6, 7(−),8(−), 9, 11-15 and 17. The method comprises the steps of:

a) Providing a spinach plant comprising an introgression fragmentobtainable from (or as in) accession NCIMB 42392 or NCIMB 42393, whichintrogression fragment confers resistance against at least Pfs races1-6, 7(−), 8(−), 9, 11-15 and 17;b) Crossing said spinach plant with another spinach plant, for examplewith a spinach plant which is susceptible against one or more ofPeronospora farinosa races 1-6, 7(−), 8(−), 9, 11-15 and 17, to produceF1 seeds;c) Optionally selfing the plants grown from F1 seeds one or more timesto produce F2, F3 or further generation selfing progeny;d) Identifying spinach plants grown from F1, F2, F3 or furthergeneration selfing progeny which have resistance against at least Pfsraces 1-6, 7(−), 8(−), 9, 11-15 and 17, and/or which comprise theintrogression fragment or a resistance-conferring part of theintrogression fragment;e) Optionally crossing said identified F1 progeny or selfing progeny tothe spinach plant of step b), to produce a backcross progeny;f) Optionally selecting backcross progeny which comprises resistanceagainst the desired Pfs races (e.g., preferably at least Pfs races 1-6,7(−), 8(−), 9, 11-15 and 17) and/or which comprise the introgressionfragment or a resistance-conferring part of the introgression fragment.

Regarding both methods, the following is encompassed herein.

In one aspect the plant of a) comprises the resistance trait as found inseeds deposited under accession number NCIMB 42392 or NCIMB 42393. Thespinach plant may be the plant grown from the seeds of the deposit orany spinach plant made using, or having used, the seed deposit and whichretains the Pfs resistance phenotype (and the gene or locus/lociconferring it). This includes commercial spinach varieties which weremade using the seed deposit. Thus, the spinach plant of a) comprises theresistance gene/locus/loci according to the invention, e.g. as found in(or as obtainable from; obtained from; derivable from; derived from)NCIMB 42392 or NCIMB 42393. The plant in a) may therefore be a plantgrown from seeds, a representative sample of which has been depositedunder NCIMB 42392 or NCIMB 42393.

Selections (or identification) in step d) and/or f) may be made based onthe phenotype (i.e. using a Pfs resistance assay) and/or based onmolecular methods, such as detection of molecular markers linked to theresistance gene or locus/loci, or other methods such as sequencing.

In a preferred embodiment, said plants according to the currentinvention comprise alleles or sequences comprising one or more of SEQ IDNOs:1-8. In some embodiments, the plants comprise one or both of SEQ IDNO:1 and SEQ ID NO:2. In some embodiments, the plants comprise one, two,three, four, five or six of SEQ ID NOs:3-8.

Preferably, said plants according to the current invention areidentifiable via use of primers directed to one or more of SEQ IDNOs:1-8, whereby the presence of at least one of these sequences or bothis a prerequisite for a plant according to the current invention. Inanother embodiment, selection of plants or progeny according to thecurrent invention occurs on the basis of the presence of at least onesequence selected from one or more of SEQ ID NOs:1-8 in said plants orprogeny. In step b) the spinach plant is, in one aspect, crossed with aspinach plant which is susceptible against at least one of the Pfs racesagainst which the plant of a) is resistant. If the second parent in b)is a spinach plant which is susceptible against at least one of the Pfsraces against which the plant of a) is resistant, then the selection instep (d) and/or (f) may be based on selecting plants which now haveresistance against that race. Steps e) and f) may be repeated one ormore times and preferably on the basis of the sequences given in one ormore of SEQ ID NOs:1-8. In some embodiments, the plants comprise one orboth of SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the plantscomprise one, two, three, four, five or six of SEQ ID NOs:3-8.

In the above methods also plants can be selected and/or identified whichretain the Pfs resistance phenotype according to the current invention,but which have a smaller introgression fragment. This can haveadvantages, as negative traits coupled to the wild introgressionfragment can thereby be removed. Initial introgression fragments fromwild sources can be quite large, e.g. 20 Mb or 30 Mb. It is thereforepreferred to reduce the size of the introgression fragment byrecombination and to select plants comprising smaller introgressionfragments, but which retain the resistance-conferring part. So, spinachwith all sizes of introgression fragments originating from (or derivedfrom; or derivable from; or obtained from; or obtainable from) seedsdeposited under accession number NCIMB 42392 or NCIMB 42393 are includedherein, as long as the Pfs resistance conferring part is retained in thespinach plant. As mentioned, the presence can be tested and selectedphenotypically and/or using molecular methods known in the art. Bypreference, selection occurs on the basis of the sequence as given inone or more of SEQ ID NOs:1-8.

Also plants obtainable or obtained by any of the above methods areembodiments of the invention. The plants according to the invention maybe any cultivated spinach, e.g. savoy, semi-savoy, flat- or smoothleaved spinach. They may be inbred lines, F1 hybrids, double haploids,transgenic plants, mutant plants, etc.

Plants of the invention can be used to generate progeny, which have orretain the Pfs resistance phenotype as obtainable from (as present in;as derivable from; as obtained or derived from) seeds deposited underaccession number NCIMB 42392 or NCIMB 42393. To generate progeny, aspinach according to the invention can be selfed and/or crossed one ormore times with another spinach plant and seeds can be collected. Thepresence of resistance according to the current invention or thegene/locus/loci responsible therefor in the progeny plants can bedetermined by the Pfs resistance phenotype and/or molecular methods,such as molecular markers (e.g. SNP markers) closely linked to the geneor locus/loci.

Also seeds from which the plants of the invention can be grown areprovided. In one embodiment, the use of a spinach plant, of whichrepresentative seeds have been deposited under accession number NCIMB42392 or NCIMB 42393, or progeny thereof (e.g. obtained by selfing) isprovided for generating a spinach plant comprising Pfs resistanceagainst one or more of Peronospora farinosa races 1-9, 11-15 and isolateUA1014APLP (Pfs 17). Preferably the spinach plant comprises resistanceagainst at least Pfs races 1-6, 7(−), 8(−), 9, 11-15 and 17.

In another embodiment, methods of use a spinach plant comprisingresistance against one or more of Peronospora farinosa races 1-6, 9,11-15 and isolate UA1014APLP (at least Pfs races 1-6, 7(−), 8(−), 9,11-15 and 17) conferred by an introgression fragment obtainable from (oras present in; as derivable from; as obtained or derived from) seedsdeposited under accession number NCIMB 42392 or NCIMB 42393, or fromprogeny thereof (e.g. obtained by selfing), is provided for generating aspinach plant comprising resistance against one or more of Peronosporafarinosa 1-9, 11-15 and isolate UA1014APLP (at least Pfs races 1-6,7(−), 8(−), 9, 11-15 and 17).

It is noted that also allelism tests can be used to determine whetherthe resistance gene in a spinach plant is the same gene/locus/loci or adifferent gene/locus/loci as the resistance gene/locus/loci as presentin NCIMB 42392 or NCIMB 42393 (or in progeny thereof). For instance,NCIMB 42392 or NCIMB 42393 (or progeny) can be crossed with anotherspinach plant comprising the same resistance phenotype and in progeny ofsuch a cross one can determine in which ratios the phenotype segregates.So in one aspect a spinach plant is provided comprising resistanceagainst one or more of P. farinosa races 1-9, 11-15 and isolateUA1014APLP (at least Pfs races 1-6, 7(−), 8(−), 9, 11-15 and 17),wherein said resistance gene/locus/loci conferring said resistancephenotype is the gene/locus/loci as present in NCIMB 42392 or NCIMB42393 (or progeny thereof), as determinable in an allelism test.Allelism tests for dominant genes are known in the art (see, e.g.,Hibberd et al., 1987, Phytopathology 77:1304-1307).

Also seeds from which any of the plants of the invention can be grownare provided, as are containers or packages containing or comprisingsuch seeds. Seeds can be distinguished from other seeds due to thepresence of the resistance gene/locus/loci, either phenotypically (basedon plants having the resistance phenotype according to the currentinvention) and/or using molecular methods.

In one aspect, seeds are packaged into small and/or large containers(e.g., bags, cartons, cans, etc.). The seeds may be pelleted prior topacking (to form pills or pellets) and/or treated with variouscompounds, such as seed coatings.

Pelleting creates round or rounded shapes, which are easily sown withmodern sowing machines. A pelleting mixture typically contains seeds andat least glue and filler material. The latter could be, for example,clay, mica, chalk or cellulose. In addition, certain additives can beincluded to improve particular properties of the pellet, e.g., a seedtreatment formulation comprising at least one insecticidal, acaricidal,nematicidal or fungicidal compound can be added directly into thepelleting mixture or in separate layers. A seed treatment formulationcan include one of these types of compounds only, a mixture of two ormore of the same type of compounds or a mixture of one or more of thesame type of compounds with at least one other insecticide, acaricide,nematicide or fungicide.

Formulations especially suitable for the application as a seed treatmentcan be added to the seed in the form of a film coating including alsothe possibility of using the coating in or on a pellet, as well asincluding the seed treatment formulation directly into the pelletmixture. Characteristically, a film coating is a uniform, dust-free,water permeable film, evenly covering the surface of all individualseeds.

Besides the formulation, the coating mixture generally also containsother ingredients such as water, glue (typically a polymer), fillermaterials, pigments and certain additives to improve particularproperties of the coating. Several coatings can be combined on a singleseed.

In addition, several combinations with film coating are possible: thefilm coating can be added on the outside of the pellet, in between twolayers of pelleting material, and directly on the seed before thepelleting material is added. Also more than 1 film coating layer can beincorporated in a single pellet. A special type of pelleting isencrusting. This technique uses less filler material, and the result isa “mini-pellet”.

Seeds may also be primed. Spinach is often primed. Priming is awater-based process that is performed on seeds to increase uniformity ofgermination and emergence from the soil, and thus enhance vegetablestand establishment. Priming decreases the time span between theemergence of the first and the last seedlings. Methods how to primespinach seeds are well known in the art.

In a further aspect plant parts, obtained from (obtainable from) a plantof the invention are provided herein, and containers or packagescomprising said plant parts. In a preferred embodiment the plant partsare leaves of spinach plants of the invention, preferably harvestedleaves, or parts of these. Leaves may be loose, bunched, fresh (e.g. inbags), frozen, blanched or boiled. Leaves may be fresh or processed,they may be part of food or feed products, such as salads, etc. Otherplant parts, of plants of the invention, include stems, cuttings,petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, roottips, protoplasts, callus, microspores, stalks, ovules, shoots, seeds,embryos, embryo sacs, cells, meristems, buds etc.

Seeds include for example seeds produced on the plant of the inventionafter self-pollination or seed produced after cross-pollination, e.g.pollination of a plant of the invention with pollen from another spinachplant or pollination of another spinach plant with pollen of a plant ofthe invention.

In a further aspect, the plant part is a plant cell. In still a furtheraspect, the plant part is a non-regenerable cell or a regenerable cell.

In another aspect the plant cell is a somatic cell. A non-regenerablecell is a cell which cannot be regenerated into a whole plant through invitro culture, but the non-regenerable cell may be in a plant or plantpart (e.g. leaves) of the invention.

In a further aspect the plant cell is a reproductive cell, such as anovule or pollen. These cells are haploid. When they are regenerated intowhole plants, they comprise the haploid genome of the starting plant. Ifchromosome doubling occurs (e.g. through chemical treatment), a doublehaploid plant can be regenerated. In one aspect the plant of theinvention, comprising the resistance gene/locus/loci is a haploid or adouble haploid spinach plant.

Moreover, there is provided an in vitro cell culture or tissue cultureof spinach plants of the invention in which the cell- or tissue cultureis derived from a plant parts described above, such as, for example andwithout limitation, leaves, pollen, embryos, cotyledon, hypocotyls,callus, meristematic cells, roots, root tips, anthers, flowers, seeds orstems, somatic cells, reproductive cells.

Also provided are spinach plants regenerated from the above-describedplant parts, or regenerated from the above-described cell or tissuecultures, said regenerated plant having a Pfs resistance phenotype, i.e.retains the resistance gene/locus/loci (or the introgression fragmentcomprising the resistance gene/locus/loci) of the invention. Theseplants can also be referred to as vegetative propagations of plants ofthe invention.

Also provided are harvested leaves of plants of the invention andpackages comprising a plurality of leaves of plants of the invention.These leaves thus comprise the resistance of the invention, detectableby e.g. linked molecular markers or phenotypically (for the originallyused whole plant and/or regenerated plant).

The invention also provides for a food or feed product comprising orconsisting of a plant part described herein. The food or feed productmay be fresh or processed, e.g., canned, steamed, boiled, fried,blanched and/or frozen etc. Examples are salad or salad mixturescomprising leaves or parts of leaves of plants of the invention.

A spinach plant of the invention or a progeny thereof retaining the Pfsresistance phenotype conferred by the gene/locus/loci and/or retainingthe introgression fragment comprising the gene/locus/loci, as present inNCIMB 42392 or NCIMB 42393, and parts of the afore-mentioned plants, canbe suitably packed for, e.g., transport, and/or sold fresh. Such partsencompass any cells, tissues and organs obtainable from the seedlings orplants, such as but not limited to: leaves, cuttings, pollen, parts ofleaves, and the like.

Leaves may be harvested immature, as baby-leaf or baby spinach, ormature. A plant, plants or parts thereof may be packed in a container(e.g., bags, cartons, cans, etc.) alone or together with other plants ormaterials. Parts can be stored and/or processed further. Encompassed aretherefore also food or feed products comprising one or more of suchparts, such leaves or parts thereof obtainable from a plant of theinvention, a progeny thereof and parts of the afore-mentioned plants.For example, containers such as cans, boxes, crates, bags, cartons,Modified Atmosphere Packaging, films (e.g. biodegradable films), etc.comprising plant parts of plants (fresh and/or processed) of theinvention are also provided herein.

In another embodiment, plants and parts of spinach plants of theinvention, and progeny of spinach plants of the invention are provided,e.g., grown from seeds, produced by sexual or vegetative reproduction,regenerated from the above-described plant parts, or regenerated fromcell or tissue culture, in which the reproduced (seed propagated orvegetatively propagated) plant comprises resistance against one or moreof Pfs races 1-9, 11-15 and isolate UA1014APLP (Pfs 17), preferably atleast Pfs races 1-6, 7(−), 8(−), 9, 11-15 and 17.

As mentioned before, whether or not a plant, progeny or vegetativepropagation comprises the Pfs resistance phenotype as conferred by thecurrent invention can be tested phenotypically using e.g. the Pfsdisease resistance assays as described above; and/or using moleculartechniques such as molecular marker analysis, DNA sequencing (e.g. wholegenome sequencing to identify the wild introgression), chromosomepainting, etc.

In one embodiment, the resistance gene/locus/loci obtainable from(obtained from; as found in) plants deposited under NCIMB 42392 or NCIMB42393, or progeny thereof, can be combined with other Peronosporafarinosa resistance genes or resistance loci or with other traits, suchresistance against bacteria (e.g. Pseudomonas syringae pv. spinacea;Erwinia carotovora), fungi (e.g. Albugo occidentalis; Colletotrichumdematium f sp. spinaciae; Stemphylium botryosum f sp. spinaciae),viruses (e.g. viruses causing curly top disease) or nematodes. This canbe done by traditional breeding techniques, e.g. by backcrossing inorder to introduce one or more traits into a plant of the invention orin order to introduce the gene/locus/loci of a plant of the inventioninto another spinach plant comprising such one or more additionaltraits. Thus, in one aspect a plant of the invention is used as a donorof the resistance according to the current invention, while in anotheraspect a plant of the invention is used as recipient of one or moreother traits.

Furthermore, the invention provides for progeny comprising or retainingthe Pfs resistance phenotype, such as progeny obtained by, e.g., selfingone or more times and/or cross-pollinating a plant of the invention withanother spinach plant of a different variety or breeding line, or with aspinach plant of the invention one or more times. In particular, theinvention provides for progeny that retain the resistancegene/locus/loci (conferring the Pfs resistance phenotype) of (as foundin) NCIMB 42392 or NCIMB 42393. In one aspect the invention provides fora progeny plant comprising the resistance according to the currentinvention, such as a progeny plant that is produced from a spinach plantcomprising the resistance according to the current invention by one ormore methods selected from the group consisting of: selfing, crossing,mutation, double haploid production or transformation.

Mutation may be spontaneous mutations or human induced mutations orsomaclonal mutations. In one embodiment, plants or seeds of theinvention may also be mutated (by e.g. irradiation, chemicalmutagenesis, heat treatment, TILLING, etc.) and/or mutated seeds orplants may be selected (e.g. natural variants, somaclonal variants,etc.) in order to change one or more characteristics of the plants.

Similarly, plants of the invention may be transformed and regenerated,whereby one or more chimeric genes are introduced into the plants.Transformation can be carried out using standard methods, such asAgrobacterium tumefaciens mediated transformation or biolistics,followed by selection of the transformed cells and regeneration intoplants.

A desired trait (e.g. genes conferring pest or disease resistance,herbicide, fungicide or insecticide tolerance, etc.) can be introducedinto the plants, or progeny thereof, by transforming a plant of theinvention or progeny thereof with a transgene that confers the desiredtrait, wherein the transformed plant retains the resistance according tothe current invention and the Pfs resistance phenotype conferred by itand contains the desired trait.

The resistance gene or locus/loci may be transferred to progeny byfurther breeding. In one aspect progeny are F1 progeny obtained bycrossing a plant of the invention with another plant or S1 progenyobtained by selfing a plant of the invention. Also encompassed are F2progeny obtained by selfing the F1 plants, or further generationprogeny. “Further breeding” encompasses traditional breeding techniques(e.g., selfing, crossing, backcrossing), marker-assisted breeding,and/or mutation breeding. In one embodiment, the progeny have the Pfsresistance phenotype of NCIMB 42392 or NCIMB 42393.

In one aspect haploid plants and/or double haploid plants of plant ofthe invention are encompassed herein, which comprise resistance againstone or more of Peronospora farinosa races 1-9, 11-15 and isolateUA1014APLP (preferably at least Pfs races 1-6, 7(−), 8(−), 9, 11-15 and17), as conferred by the gene/locus/loci or by the introgressionfragment comprising the resistance gene. Haploid and double haploid (DH)plants can for example be produced by anther or microspore culture andregeneration into a whole plant. For DH production chromosome doublingmay be induced using known methods, such as colchicine treatment or thelike. So, in one aspect a spinach plant is provided, comprising Pfsresistance phenotype as described, wherein the plant is a double haploidplant.

In another embodiment the invention relates to a method for producingspinach seed, comprising crossing a plant of the invention with itselfor a different spinach plant and harvesting the resulting seed. In afurther embodiment the invention relates to seed produced according tothis method and/or a spinach plant produced by growing such seed. Thus,a plant of the invention may be used as male and/or female parent, inthe production of spinach seeds, whereby the plants grown from saidseeds comprise resistance against one or more of Peronospora farinosaraces 1-9, 11-15 and isolate UA1014APLP (preferably at least Pfs races1-6, 7(−), 8(−), 9, 11-15 and 17).

Thus, in one aspect progeny of a spinach plant of the invention areprovided, wherein the progeny plant is produced by selfing, crossing,mutation, double haploid production or transformation and wherein theprogeny retain the resistance gene/locus/loci (and phenotype conferredby it) described herein, i.e. obtainable by crossing a spinach plant,grown from seeds deposited under accession number NCIMB 42392 or NCIMB42393, with another spinach plant. In other words, the resistance geneor locus (or introgression fragment comprising the gene or locus)present in or as derivable from seed deposited as NCIMB 42392 or NCIMB42393 is retained in the progeny plants.

Molecular markers may also be used to aid in the identification of theplants (or plant parts or nucleic acids obtained therefrom) containingthe resistance gene or locus or allele(s). For example, one can developone or more suitable molecular markers which are closely genetically(and preferably also physically) linked to the resistance gene, locus orallele. This can be done by crossing a resistant spinach plant with asusceptible spinach plant and developing a segregating population (e.g.F2 or backcross population) from that cross. The segregating populationcan then be phenotyped for Pfs resistance and genotyped using e.g.molecular markers such as SNPs (Single Nucleotide Polymorphisms), AFLPs(Amplified Fragment Length Polymorphisms; see, e.g. EP 534 858), orothers, and by software analysis molecular markers which co-segregatewith the Pfs resistance trait in the segregating population can beidentified and their order and genetic distance (centimorgan distance,cM) to the resistance gene or locus can be identified. Molecular markerswhich are closely linked to resistance locus or loci, e.g. markers at a5 cM distance or less, can then be used in detecting and/or selectingplants (e.g. plants of the invention or progeny of a plant of theinvention) or plant parts comprising or retaining the introgressionfragment comprising the resistance gene or locus. Such closely linkedmolecular markers can replace phenotypic selection (or be used inaddition to phenotypic selection) in breeding programs, i.e. in MarkerAssisted Selection (MAS). Preferably flanking markers are used in MAS,i.e. one marker on either side of the resistance gene or locus/loci.

Any other type of molecular marker and/or other assay that is able toidentify the relative presence or absence of a trait of interest in aplant or plant part can also be useful for breeding purposes.

Said progeny plants according to the current invention comprise allelesor sequences which comprise one or more of SEQ ID NOs:1-8. In someembodiments, the progeny plants comprise one or both of SEQ ID NO:1 andSEQ ID NO:2. In some embodiments, the progeny plants comprises one, two,three, four, five or six of SEQ ID NOs:3-8.

Preferably said progeny plants according to the current invention areidentifiable via use of primers directed to one or more of SEQ IDNOs:1-8, whereby the presence of at least one of these sequences is aprerequisite for a plant according to the current invention. In someembodiments, selection of plants according to the current inventionoccurs on the basis of the presence of at least one sequence selectedfrom SEQ ID NOs:1-8. In some embodiments, the plants comprise one orboth of SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the plantscomprises one, two, three, four, five or six of SEQ ID NOs:3-8.

Sequence analysis of SEQ ID NOs:1-8 was performed using the BLASTn toolfound on the “spinachbase” website in comparison to the draft genome ofSpinacia oleracea cultivar Sp75′, released February 2017. All eightsequences were found to align with sequences of chromosome 3 of ‘Sp75’,which has a length of approximately 113 megabases.

TABLE I Alignment with the genome of Spinacia oleracea cultivar ‘Sp75’SEQ ID NO: Approximate location on ‘Sp75’ chromosome 3 1 1,271,971 bp 21,264,341 bp 3 3,861,266 bp 4 3,963,668 bp 5 3,963,678 bp 6 3,965,092 bp7 4,005,190 bp 8 4,005,208 bp

Deposit Information

A total of 2500 seeds of spinach line ‘B11-523-1-17’ described inExample 1 were deposited under accession number NCIMB 42392 by PopVriend Seeds on 7 Apr. 2015, at the NCIMB Ltd., Ferguson Building,Craibstone Estate, Bucksbum, Aberdeen AB21 9YA, United Kingdom (NCIMB).Likewise, a total of 2500 seeds of spinach line ‘B11-505-3-17’ describedin Example 2 were deposited under accession number NCIMB 42393 by PopVriend Seeds on 7 Apr. 2015, at NCIMB. Subject to 37 C.F.R. § 1.808(b),all restrictions imposed by the depositor on the availability to thepublic of the deposited material will be irrevocably removed upon thegranting of the patent. The deposit will be maintained for a period of30 years, or 5 years after the most recent request or for theenforceable life of the patent whichever is longer, and will be replacedif it ever becomes nonviable during that period. Applicant does notwaive any rights granted under this patent on this application or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

Enumberated Embodiments

The following enumerated embodiments are representative of some aspectsof the invention.

1. A spinach plant comprising resistance against Peronospora farinosaraces 1-9, 11-15 and isolate UA1014APLP (Pfs 17), or against Pfs races1-6, 7(−), 8(−), 9, 11-15 and 17.2. The spinach plant of embodiment 1, wherein the plant is obtainable byintrogression from a plant grown from seeds of which a representativesample has been deposited with NCIMB under accession number NCIMB 42392or NCIMB 42393.3. The spinach plant of embodiment 1 or 2, whereby said spinach plantcomprises at least one allele or sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2; or selected from the groupconsisting of SEQ ID NOs:3-8.4. The spinach plant of embodiment 3, whereby said allele or sequence islinked to the resistance.5. The spinach plant of any one of embodiments 1 to 4, whereby saidspinach plant is a hybrid plant.6. The spinach plant of any one of embodiments 1 to 4, whereby saidspinach plant is an inbred plant.7. The spinach plant of any one of embodiments 1 to 6, whereby saidplant is selected from the group consisting of: savoy, semi-savoy, flator smooth leaved.8. A progeny plant of the spinach plant of any one of embodiments 1 to7, wherein said progeny plant retains resistance against Peronosporafarinosa races 1-9, 11-15, and isolate UA1014APLP (Pfs 17), or againstPfs races 1-6, 7(−), 8(−), 9, 11-15 and 17.9. The progeny plant of embodiment 8, wherein the progeny plant isproduced by one or more methods selected from the group consisting of:selfing, crossing, mutation, double haploid production ortransformation.11. The progeny plant of embodiment 8 or 9, whereby said progeny plantcomprises at least one allele or sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2; or selected from the groupconsisting of SEQ ID NOs:3-8.12. Seed from which a spinach plant of any one of embodiments 1 to 7 canbe grown.13. Use of one or more seeds as deposited under accession number NCIMB42392 or NCIMB 42392 or progeny thereof for generating a spinach plantcomprising resistance against Peronospora farinosa races 1-9, 11-15 andisolate UA1014APLP (Pfs 17), or against Pfs races 1-6, 7(−), 8(−), 9,11-15 and 17.14. A part of the spinach plant of any one of embodiments 1 to 7 or apart of the progeny plant of any one of embodiments 8 to 10, wherein thepart is selected from the group consisting of: stems, cuttings,petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, roottips, protoplasts, callus, microspores, stalks, ovules, shoots, seeds,embryos, embryo sacs, cells, meristems, buds, leaves15. A cell culture or tissue culture comprising cells or tissue derivedfrom the part of embodiment 13.16. A spinach plant regenerated from the cell or tissue culture ofembodiment 14, wherein said spinach plant comprises resistance againstPeronospora farinosa races 1-9, 11-15 and isolate UA1014APLP (Pfs 17) oragainst Pfs races 1-6, 7(−), 8(−), 9, 11-15 and 17.17. The spinach plant of embodiment 15, wherein said spinach plantcomprises at least one allele or sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:2; or selected from the groupconsisting of SEQ ID NOs:3-8.18. A food product comprising the harvested leaves of the plant of anyone of embodiments 1 to 7 or 15 to 16 or a progeny according to any ofthe claims 8 to 10.19. A container comprising one or more spinach plants of any one ofembodiments 1, 15 or 8, in a growth substrate for harvest of leaves fromsaid plants.20. A method for generating a spinach plant comprising resistanceagainst a plurality of Peronospora farinosa f. sp. spinaciae (Pfs)races, comprising the steps of:

a) providing a spinach plant comprising an introgression fragmentobtainable from accession number NCIMB 42392 or NCIMB 42393, whichintrogression fragment confers resistance against the plurality of Pfsraces;

b) crossing said spinach plant with another spinach plant, which issusceptible against one or more of the plurality of Pfs races;

c) optionally selfing the plants grown from F1 seeds one or more timesto produce F2, F3 or further generation selfing progeny;

d) identifying spinach plants grown from F1, F2, F3 or furthergeneration selfing progeny which have resistance against the pluralityof Pfs races and/or which comprise the introgression fragment or aresistance-conferring part of the introgression fragment;

e) optionally crossing said identified F1 progeny or selfing progeny tothe spinach plant of step b), to produce a backcross progeny;

f) optionally selecting backcross progeny which comprises resistance theplurality of Pfs races and/or which comprise the introgression fragmentor a resistance-conferring part of the introgression fragment,

wherein the plurality of Pfs races comprises Pfs races 1-9, 11-15 andisolate UA1014APLP (Pfs 17) or Pfs races 1-6, 7(−), 8(−), 9, 11-15 and17.21. The method of embodiment 19, whereby said identification step instep d) occurs via marker assisted selection.22. The method of embodiment 19, whereby said identifying spinach plantsand/or selecting backcross progeny occurs by identifying the presence ofat least one allele or sequence in said plants or progeny; wherein theat least one allele or sequence is selected from the group consisting ofSEQ ID NO:1 and SEQ ID NO:2; or selected from the group consisting ofSEQ ID NOs:3-8.23. A spinach plant comprising resistance against Peronospora farinosa(Pfs) race 14, wherein said resistance is conferred by a resistancegene, which is present in seeds deposited under accession number NCIMB42392 or NCIMB 42393.24. The plant of embodiment 23, wherein said spinach plant furthercomprises resistance against Pfs races 1-6.25. The plant of embodiment 23, wherein said spinach plant furthercomprises resistance against Pfs isolate UA1014APLP.26. The plant of embodiment 23, wherein said spinach plant comprisesresistance against Pfs races 11-15 and isolate UA1014APLP.27. The plant of embodiment 23, wherein said spinach plant comprises atleast one allele selected from the group consisting of:

a ‘T’ allele at a single nucleotide polymorphism (SNP) at position 51 ofSEQ ID NO:1;

a ‘T’ allele at a SNP of position 51 of SEQ ID NO:2;

a ‘C’ allele at the SNP of position 51 of SEQ ID NO:3;

a ‘T’ allele at the SNP of position 51 of SEQ ID NO:4;

a ‘G’ allele at the SNP of position 51 of SEQ ID NO:5;

a ‘C’ allele at the SNP of position 51 of SEQ ID NO:6;

a ‘C’ allele at the SNP of position 51 of SEQ ID NO:7; and

a ‘C’ allele at the SNP of position 51 of SEQ ID NO:8.

28. The plant of embodiment 27, wherein said spinach plant comprises atleast one nucleotide sequence selected from the group consisting of SEQID NOs:1-8, or at least one nucleotide sequence having at least 90%identity to one ore more of the group consisting of SEQ ID NOs:1-8.

29. A seed from which the plant of embodiment 23 can be grown.

30. A leaf of the plant of embodiment 23.

31. A progeny plant of the plant of embodiment 23, wherein said progenyplant retains the resistance gene which confers resistance to Pfs race14.

32. The progeny plant of embodiment 31, wherein said progeny plant isresistant to Pfs races 11-15 and isolate UA1014APLP.

33. The progeny plant of embodiment 31, wherein said progeny plant isproduced by one or more methods of selfing, crossing, mutation, doublehaploid production or transformation.

34. The progeny plant of embodiment 31, wherein said progeny plantcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1-8, or at least one nucleotide sequence havingat least 90% identity to one ore more of the group consisting of SEQ IDNOs:1-8.

35. A method of generating a spinach plant comprising resistance againstPfs race 14, comprising growing a plant from a seed deposited underaccession number accession number NCIMB 42392 or NCIMB 42393 or aprogeny thereof, wherein said progeny comprises a resistance gene whichconfers resistance to Pfs race 14, wherein the resistance gene ispresent in seeds deposited under accession number NCIMB 42392 or NCIMB42393.

36. The method of embodiment 35, wherein said resistance gene confersresistance against Pfs races 11-15 and isolate UA1014APLP.

37. A part of the spinach plant of embodiment 23, wherein the part isselected from the group consisting of a stem, a cutting, a petiole, acotyledon, a flower, an anther, a pollen, an ovary, a root, a root tip,a protoplast, a callus, a microspore, a stalk, an ovule, a shoot, aseed, an embryo, an embryo sac, a cell, a meristem, a bud or a leaf.

38. A cell culture or tissue culture comprising a cell or a tissuederived from the part of embodiment 37.

39. A spinach plant regenerated from the cell culture or tissue cultureof embodiment 38 and comprising resistance against Pfs race 14, whereinsaid resistance is conferred by a resistance gene present in seedsdeposited under accession number NCIMB 42392 or NCIMB 42393.

40. The spinach plant of embodiment 39, wherein said spinach plantcomprises at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1-8, or at least one nucleotide sequence havingat least 90% identity to one ore more of the group consisting of SEQ IDNOs:1-8.

41. A food product comprising harvested leaves of the spinach plant ofembodiment 23.

42. A container comprising the spinach plant of embodiment 23, in agrowth substrate for harvest of leaves from said plant.

EXAMPLES

The present disclosure will be more fully understood by reference to thefollowing examples. It should not, however, be construed as limiting thescope of the present disclosure. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Downy Mildew-Resistant Spinach Line ‘B11-523-1-17’ Origin ofBreeding

Seeds of spinach line ‘B11-523-1-17’ were deposited as accession numberNCIMB 42392. The ‘B11-523-1-17’ spinach line was developed by firstcrossing a plant of variety ‘Viroflay’ (S. oleracea) with a plant of S.turkestanica with accession number 32. The final outcome was a spinachplant with resistance against Pfs races 1-6, 7(−), 8(−), 9, 11-15 andisolate UA1014APLP (Pfs 17). Seeds of this final outcome were depositedas accession number NCIMB 42392. Observation during the varietyselections confirmed that ‘B11-523-1-17’ is uniform and stable.

TABLE 1-1 Characteristics of the ‘B11-523-1-17’ Spinach LineCharacteristic ‘B11-523-1-17’ Seed: spines absent Plant: red colorationof stem, petioles absent and veins Leaf Blade: intensity of green colordark Leaf Blade: blistering absent or very weak Petiole: length longLeaf Blade: shape medium ovate Leaf Blade: shape of apex obtuseProportion of monoecious plants very high Time of start of boltingbetween early and medium

The crossing scheme was as follows:

Step 1: crossing between cultivar ‘Viroflay’ (S. oleracea) and S.turkestanica accession number 32;

Step 2: crossing of F1 progeny plants from step 1 with a S. oleraceacultivar with internal reference ‘PV1372’;

Step 3: selfing of the plants obtained from step 2 after selection fordowny mildew resistance; and

Step 4: selfing of the plants obtained from step 3 after selection fordowny mildew resistance.

Plants from seeds resulting from step 4 were grown and downy mildew testresults showed resistance against Pfs races 1-6, 7(−), 8(−), 9, 11-15and isolate UA1014APLP (Pfs 17). The presence of alleles/sequences asgiven in SEQ ID NO:1 and SEQ ID NO:2 was confirmed. ‘B11-523-11’ is aline of the F2 generation, and ‘B11-523-11-17’ is a line of the F3generation.

In accordance with the disclosure, novel varieties may be created bycrossing plants grown from NCIMB 42392 followed by multiple generationsof breeding according to well-known methods. New varieties may becreated by crossing with any second plant. Once initial crosses havebeen made, inbreeding and selection take place to produce new varieties.For development of a uniform line, often five or more generations ofselfing and selection are involved. It is supposed that the presentdisclosure is not restricted to any form of realization describedpreviously and that some modifications can be added to the presentedexample of fabrication without reappraisal of the appended claims.

Mapping the Resistance

A mapping population was created as described in the “Origin ofbreeding” section. In brief, F1 progeny of the cross of between thecultivar ‘Viroflay’ (S. oleracea) and S. turkestanica accession number32 was created with internal reference ‘B11-523’. The outcome was aspinach plant with resistance against Pfs races 1-9, 11-15 and isolateUA1014APLP. An F2 population was obtained by crossing a ‘B11-523’individual with a S. oleracea cultivar with internal reference ‘PV1372’.Approximately 120 plants were tested for downy mildew resistance againststrain Pfs 14. The parents, the F1 progenitor used and 96 randomlychosen F2 individuals for which the phenotypic data were analyzed wereused for molecular genetic analysis.

TABLE 1-2 Pfs Resistance of 96 F2 Plants Pfs 14 Number PercentageResistant 55 57% Susceptible 41 43%

Genomic DNA of the parents and F2 progeny was isolated and the F2progeny was pooled in resistant and susceptible bulks. The gDNA wasprepared for Illumina HiSeq sequencing. Briefly, the gDNA was preparedfor shot gun library preparation by strict fragmentation and end repairof gDNA, adapter ligation, size selection (approximately 300 bp) PCRamplification, library purification and Quality Control. Two flowchannels were prepared and the libraries were sequenced in Hiseq25002×150 bp paired-end mode. The data was collected and filtered accordingto Quality scores in Illumina pipeline 1.8.

The reference sequences of S. oleracea cultivar ‘Viroflay’ were obtainedfrom the Spinach Genome Sequence Resources (SGSR, UC Davis GenomeCenter, Davis, USA) and is namedspinach_assembly-repeatdetect_PACBIO_V1.3. The reference sequencescontain 2882 high quality contigs with an average length of 316,211,89nucleotides. The obtained reads of the parents were stringently mappedusing CLC Genomics Workbench V. 9.5.1 with the following parameters:

References=spinach_assembly-repeatdetect_PACBIO_V1.3

Masking mode=No masking

Match score=1

Mismatch cost=2

Cost of insertions and deletions=Linear gap cost

Insertion cost=3

Deletion cost=3

Length fraction=0.8

Similarity fraction=0.95

Global alignment=No

Auto-detect paired distances=Yes

Non-specific match handling=Map randomly

Output mode=Create stand-alone read mappings

Create report=Yes

Collect un-mapped reads=No

TABLE 1-3 Read Mapping Statistics of the Internal Reference Variety‘PV1372’ Percentage Number Percentage ‘PV1372’ Count of reads of basesof bases Reference 2,882 — 911,322,679 — Mapped reads 73,862,266 94.78%11,079,339,900 94.78% Not mapped 4,069,614 5.22% 610,442,100 5.22% readsReads in pairs 64,277,672 82.48% 9,641,650,800 82.48% Broken paired9,584,594 12.30% 1,437,689,100 12.30% reads Total reads 77,931,880100.00% 11,689,782,000 100.00%

TABLE 1-4 Read Mapping Statistics of ‘B11-523-1’ Percentage Percentage‘B11-523-1’ Count of reads Number of bases of bases Reference 2,882 —911,322,679 — Mapped reads 117,314,539 95.55% 17,597,180,850 95.55% Notmapped 5,466,625 4.45% 819,993,750 4.45% reads Reads in pairs101,964,742 83.05% 15,294,711,300 83.05% Broken paired 15,349,797 12.50%2,302,469,550 12.50% reads Total reads 122,781,164 100.00%18,417,174,600 100.00%

The ‘B11-523’ mapping file was used in Probabilistic Variant Detection.The Variant data file was used to filter for variants that are alsopresent in the ‘PV1372’ mapping file with various settings to optimizethe data. A total of 14,897 variants (Single Nucleotide Variants orSNVs, and insertions/deletions) were detected for the ‘PV1372’ mappingfile and a total of 209,506 variants were detected for the ‘B11-523’mapping file.

The variant file of ‘B11-523’ was both in silico and manually filteredfor: Known variants in the ‘PV1372’ mapping file, Type (SNV), Frequencyof the variant, Coverage, Control coverage, Probability, Forward/reversebalance, Average base quality and the Reference allele. In total, 96heterozygous SNVs were obtained. The contigs containing the variantswere manually checked for heterozygous/homozygous coverage of the readmappings and rejected when both frequencies were in vicinity of eachother in a random fashion. It is anticipated that S. turkestanica readmappings are semi-hemizygous (i.e., blocks of hemizygous regions areflanked by regions with heterozygosity) as has been seen for previouscrossings with genomes having high scores of polymorphism at thenucleotide level. Therefore, a number of dominant SNVs for thehemizygous regions of the aforementioned contigs were also selected. Atotal of 48 SNVs of eight different contigs were selected for KASPassays on the 96 F2 individuals. Two SNPs were found to be associatedwith the phenotypic data (e.g., Pfs 14 resistance. These SNPs can beused for the identification of plants having the Pfs resistancephenotype according to the current disclosure (see also sequences givenin the sequence listing below). More in detail, plants according to thecurrent invention are linked to a SNP on position 137372 (C to T) and/ora SNP on position 144376 (A to T).

TABLE 1-5 SNPs Identified in ‘B11-523’ Associated with Pfs 14 ResistanceContig Region Reference Allele Count Coverage Forward/reverse balanceuti_cns_0000548 137,372 C T 13 23 0.5 uti_cns_0000548 144,376 A T 11 240.43

SEQ ID NO:1=Nucleotide sequence of ‘B11-523’ containing a ‘T’ allele atthe SNP of position 51 (region 137,372 of ut_cns_0000548):

GAATTAAGAATTGGTGTTGGATTTTACTCTGTAGTTTTCAGGTATATCGTTAGGGATGCAAATTAGTCGAGACTTTCAAGAATTGATCTTGGAGTATTTG G.

SEQ ID NO:2=Nucleotide sequence of ‘B11-523’ containing a ‘T’ allele atthe SNP of position 51 (region 144,376 of ut_cns_0000548):

CTTTGTTAATTATATTCATGCTATTGCGCTCTATATTAAAGCTAAGAACATGAGGTGTTACAAGATGCCATGTTCCGGTCGATCGTTACAAATTGGATTA A.

An example of a read mapping of B11-523 on ‘Viroflay’ contiguti_cns_0000548 containing heterozygous informative SNPs of position137,372 (C to T) can be found in FIG. 1 of WO 2017/081187, which isincorporated by reference.

Example 2: Downy Mildew-Resistant Spinach Line ‘B11-505-3-17’ Origin ofBreeding

Seeds of spinach line ‘B11-505-3-17’ were deposited as accession numberNCIMB 42393. The ‘B11-505-3-17’ spinach line was developed by firstcrossing a plant of variety ‘Viroflay’ (S. oleracea) with a plant of S.turkestanica with accession number KK28. The final outcome was a spinachplant with resistance against Pfs races 1-6, 7(−), 8(−), 9, 11-15 andisolate UA1014APLP (Pfs 17). Seeds of this final outcome were depositedas accession number NCIMB 42393. Observation during the varietyselections confirmed that ‘B11-505-3-17’ is uniform and stable.

The crossing scheme was as follows:

Step 1: crossing between cultivar ‘Viroflay’ (S. oleracea) and S.turkestanica accession number KK28;

Step 2: crossing of F1 progeny plants from step 1 with a S. oleraceacultivar ‘Crocodile’;

Step 3: selfing of the plants obtained from step 2 after selection fordowny mildew resistance; and

Step 4: selfing of the plants obtained from step 3 after selection fordowny mildew resistance.

Plants from seeds resulting from step 4 were grown and downy mildew testresults showed resistance against Pfs races 1-6, 7(−), 8(−), 9, 11-15and isolate UA1014APLP. Presence of alleles/sequences as given in SEQ IDNOs:3-8 was confirmed. B11-505-3 a line of the F2 generation, andB11-505-3-17 a line of the F3 generation.

In accordance with the disclosure, novel varieties may be created bycrossing plants grown from NCIMB 42393 followed by multiple generationsof breeding according to well-known methods. New varieties may becreated by crossing with any second plant. Once initial crosses havebeen made, inbreeding and selection take place to produce new varieties.For development of a uniform line, often five or more generations ofselfing and selection are involved. It is supposed that the presentdisclosure is not restricted to any form of realization describedpreviously and that some modifications can be added to the presentedexample of fabrication without reappraisal of the appended claims.

TABLE 2-1 Characteristics of the ‘B11-505-3-17’ Spinach LineCharacteristic ‘B11-505-3-17’ Seed: spines absent Plant: red colorationof stem, petioles and veins absent Leaf Blade: intensity of green colordark Leaf Blade: blistering absent or very weak Petiole: length mediumLeaf Blade: shape medium ovate Leaf Blade: shape of apex obtuseProportion of monoecious plants very high Time of start of bolting early

Mapping of the Resistance

A mapping population was created as described in the “Origin ofbreeding” section. In brief, F1 progeny of the cross of between thecultivar ‘Viroflay’ (S. oleracea) and S. turkestanica accession numberKK28 was created and given internal reference ‘B11-505’. An F2population was obtained by crossing a ‘B11-505’ individual with a S.oleracea cultivar ‘Crocodile’. Approximately 120 plants were tested fordowny mildew resistance against strain Pfs 14. The parents, the F1progenitor used and 96 randomly chosen F2 individuals for which thephenotypic data were analyzed were used for molecular genetic analysis.

Genomic DNA of the parents and F2 progeny was isolated and the F2progeny was pooled in resistant and susceptible bulks. The gDNA wasprepared for Illumina HiSeq sequencing. Briefly, the gDNA was preparedfor shotgun library preparation by strict fragmentation and end repairof gDNA, adapter ligation, size selection (approximately 300 bp) PCRamplification, library purification and Quality Control. Two flowchannels were prepared and the libraries were sequenced in Hiseq25002×150 bp paired-end mode. The data was collected and filtered accordingto Quality scores in Illumina pipeline 1.8.

The reference sequences of S. oleracea cultivar ‘Viroflay’ were obtainedfrom the Spinach Genome Sequence Resources (SGSR, UC Davis GenomeCenter, Davis, USA) and is namedspinach_assembly-repeatdetect_PACBIO_V1.3. The reference sequencescontain 2882 high quality contigs with an average length of 316,211,89nucleotides. The obtained reads of the parents were stringently mappedusing CLC Genomics Workbench V. 9.5.1 with the following parameters:

References=spinach_assembly-repeatdetect_PACBIO_V1.3

Masking mode=No masking

Match score=1

Mismatch cost=2

Cost of insertions and deletions=Linear gap cost

Insertion cost=3

Deletion cost=3

Length fraction=0.8

Similarity fraction=0.95

Global alignment=No

Auto-detect paired distances=Yes

Non-specific match handling=Map randomly

Output mode=Create stand-alone read mappings

Create report=Yes

Collect un-mapped reads=No

TABLE 2-1 The read mapping statistics of internal reference variety‘Crocodile’ Percentage Percentage ‘Crocodile’ Count of reads Number ofbases of bases References 2,882 — 911,322,679 — Mapped reads 82,525,10896.58% 12,378,766,200 96.58% Not mapped 2,920,510 3.42% 438,076,5003.42% reads Reads in pairs 72,778,792 85.18% 10,916,818,800 85.18%Broken paired 9,746,316 11.41% 1,461,947,400 11.41% reads Total reads85,445,618 100.00% 12,816,842,700 100.00%

TABLE 2-2 The read mapping statistics of ‘B11-505’ Percentage Percentage‘B11-505’ Count of reads Number of bases of bases References 2,882 —911.322.679 — Mapped reads 120,355,960 95.97% 18,053,394,000 95.97% Notmapped 5,059,938 4.03% 758,990,700 4.03% reads Reads in pairs104,653,714 83.45% 15,698,057,100 83.45% Broken paired 15,702,246 12.52%2,355,336,900 12.52% reads Total reads 125,415,898 100.00%18,812,384,700 100.00%

The ‘B11-505’ mapping file was used in Probabilistic Variant Detection.The Variant data file was used to filter for variants that are alsopresent in the ‘Crocodile’ mapping file with various settings tooptimize the data. A total of 57,394 variants (Single NucleotideVariants, or SNVs, and insertions/deletions) were detected for the‘Crocodile’ mapping file and a total of 446,034 variants were detectedfor the ‘B11-505’ mapping file.

The variant file of ‘B11-505’ was both in silico and manually filteredfor: Known variants in the ‘Crocodile’ mapping file, Type (SNV),Frequency of the variant, Coverage, Control coverage, Probability,Forward/reverse balance, Average base quality and the Reference allele.In total, 157 heterozygous SNVs were obtained. The contigs containingthe variants were manually checked for heterozygous/homozygous coverageof the read mappings and rejected when both frequencies were in vicinityof each other in a random fashion. It is anticipated that S.turkestanica read mappings are semi-hemizygous (i.e., blocks ofhemizygous regions are flanked by regions with heterozygosity) as hasbeen seen for previous crossings with genomes having high scores ofpolymorphism at the nucleotide level. Therefore, a number of dominantSNVs for the hemizygous regions of the aforementioned contigs were alsoselected. A total of 48 SNVs of eight different contigs were selectedfor KASP assays on the 96 F2 individuals. Six regions were found to beassociated with the phenotypic data (e.g., Pfs 14 resistance). TheseSNPs can be used for the identification of plants having the Pfsresistance phenotype according to the current disclosure (see alsosequences given in the sequence listing below).

TABLE 2-3 SNPs Identified in ‘B11-505’ Associated with Pfs 14 ResistanceContig Region Reference Allele Count Coverage Forward/reverse balanceuti_cns_0000042 58,929 T C 11 25 0.43 uti_cns_0000042 169,323 A T 11 250.5 uti_cns_0000042 169,333 A G 13 26 0.5 uti_cns_0000042 170,880 G C 1126 0.43 uti_cns_0000042 342,263 T C 15 29 0.42 uti_cns_0000042 342,281 TC 15 29 0.44

SEQ ID NO:3=Nucleotide sequence of ‘B11-505’ containing a ‘C’ allele atthe SNP of position 51 (region 58,929 of uti_cns_000042):

AGCGAAGAGGTTTTAAGAGCTATTTTGATTTAGTCCATTATATTCAATGCCGAAAAGAACAAATGTATCTTTGGCTCCATCGGAAATGGACAGATTTAGA T.

SEQ ID NO:4=Nucleotide sequence of ‘B11-505’ containing a ‘T’ allele atthe SNP of position 51 (region 169,323 of uti_cns_000042):

AATCTTCTGCGGCTTCTGCCGAAGCTCTAGTTTGTTTTATTTGAGGTTCTTTAACAGCATATTCCCCAGCCAGAGAAGGAAGCGGCTGTTGAGTTTCTTG A.

SEQ ID NO:5=Nucleotide sequence of ‘B11-505’ containing a ‘G’ allele atthe SNP of position 51 (region 169,333 of uti_cns_000042):

GGCTTCTGCCGAAGCTCTAGTTTGTTTTATTTGAGGTTCTATAACAGCATGTTCCCCAGCCAGAGAAGGAAGCGGCTGTTGAGTTTCTTGATGCAACTGA C.

SEQ ID NO:6=Nucleotide sequence of ‘B11-505’ containing a ‘C’ allele atthe SNP of position 51 (region 170,880 of uti_cns_000042):

ACAACACAACTAAATTCGACCTAACAGTCCCTAACAAACCAGAATCGTTACAATAATCAGACGAATCCCAACAATAATCATGAATTACCTGCTTAATCGA G.

SEQ ID NO:7=Nucleotide sequence of ‘B11-505’ containing a ‘C’ allele atthe SNP of position 51 (region 342,263 of uti_cns_000042):

TTTAGAAACTGAGAAAATAAAGATTTTGATGTTTCCATCCCTAATGTAACCGCTGCAAGAGATCTGAATAGTATGCAGAATGTTGAGCAGAACCCAATTA T.

SEQ ID NO:8=Nucleotide sequence of ‘B11-505’ containing a ‘C’ allele atthe SNP of position 51 (region 342,281 of uti_cns_000042):

AAAGATTTTGATGTTTCCATCCCTAATGTAACTGCTGCAAGAGATCTGAACAGTATGCAGAATGTTGAGCAGAACCCAATTATGAGGTAAACTGTAATCA A.

An example of a read mapping of B11-505 on ‘Viroflay’ contiguti_cns_0000042 containing heterozygous informative SNPs of position169,333 (A to G) can be found in FIG. 1 of WO 2017/081189, which isincorporated by reference.

What is claimed is:
 1. A spinach plant comprising resistance against Peronospora farinosa (Pfs) race 14, wherein said resistance is conferred by a resistance gene, which is present in seeds deposited under accession number NCIMB 42392 or NCIMB
 42393. 2. The plant of claim 1, wherein said spinach plant further comprises resistance against Pfs races 1-6.
 3. The plant of claim 1, wherein said spinach plant further comprises resistance against Pfs isolate UA1014APLP.
 4. The plant of claim 1, wherein said spinach plant comprises resistance against Pfs races 11-15 and isolate UA1014APLP.
 5. The plant of claim 1, wherein said spinach plant comprises at least one allele selected from the group consisting of: a ‘T’ allele at a single nucleotide polymorphism (SNP) at position 51 of SEQ ID NO:1; a ‘T’ allele at a SNP of position 51 of SEQ ID NO:2; a ‘C’ allele at the SNP of position 51 of SEQ ID NO:3; a ‘T’ allele at the SNP of position 51 of SEQ ID NO:4; a ‘G’ allele at the SNP of position 51 of SEQ ID NO:5; a ‘C’ allele at the SNP of position 51 of SEQ ID NO:6; a ‘C’ allele at the SNP of position 51 of SEQ ID NO:7; and a ‘C’ allele at the SNP of position 51 of SEQ ID NO:8.
 6. The plant of claim 5, wherein said spinach plant comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOs:1-8, or at least one nucleotide sequence having at least 90% identity to one ore more of the group consisting of SEQ ID NOs:1-8.
 7. A seed from which the plant of claim 1 can be grown.
 8. A leaf of the plant of claim
 1. 9. A progeny plant of the plant of claim 1, wherein said progeny plant retains the resistance gene which confers resistance to Pfs race
 14. 10. The progeny plant of claim 9, wherein said progeny plant is resistant to Pfs races 11-15 and isolate UA1014APLP.
 11. The progeny plant of claim 9, wherein said progeny plant is produced by one or more methods of selfing, crossing, mutation, double haploid production or transformation.
 12. The progeny plant of claim 9, wherein said progeny plant comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOs:1-8, or at least one nucleotide sequence having at least 90% identity to one ore more of the group consisting of SEQ ID NOs:1-8.
 13. A method of generating a spinach plant comprising resistance against Pfs race 14, comprising growing a plant from a seed deposited under accession number accession number NCIMB 42392 or NCIMB 42393 or a progeny thereof, wherein said progeny comprises a resistance gene which confers resistance to Pfs race 14, wherein the resistance gene is present in seeds deposited under accession number NCIMB 42392 or NCIMB
 42393. 14. The method of claim 13, wherein said resistance gene confers resistance against Pfs races 11-15 and isolate UA1014APLP.
 15. A part of the spinach plant of claim 1, wherein the part is selected from the group consisting of a stem, a cutting, a petiole, a cotyledon, a flower, an anther, a pollen, an ovary, a root, a root tip, a protoplast, a callus, a microspore, a stalk, an ovule, a shoot, a seed, an embryo, an embryo sac, a cell, a meristem, a bud or a leaf.
 16. A cell culture or tissue culture comprising a cell or a tissue derived from the part of claim
 15. 17. A spinach plant regenerated from the cell culture or tissue culture of claim 16 and comprising resistance against Pfs race 14, wherein said resistance is conferred by a resistance gene present in seeds deposited under accession number NCIMB 42392 or NCIMB
 42393. 18. The spinach plant of claim 17, wherein said spinach plant comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOs:1-8, or at least one nucleotide sequence having at least 90% identity to one ore more of the group consisting of SEQ ID NOs:1-8.
 19. A food product comprising harvested leaves of the spinach plant of claim
 1. 20. A container comprising the spinach plant of claim 1, in a growth substrate for harvest of leaves from said plant. 